The present invention relates in general to agents and methods for treating tumor cell growth progression and metastasis, and more particularly to combinations of an inhibitor of inducible nitric oxide synthase and an inhibitor of cylcooxygenase-2 for chemoprevention and treatment of cancers and methods of using the agents in medicine.
Cancers continue to be a leading cause of death in developed nations, and in particular colorectal cancer is one of the leading causes of cancer deaths in both men and women in Western countries, including the United States (Landis et al. (2000) CA-A Cancer J. Clinicians, 48: 6-27.).
Epidemiological and experimental studies indicate that the risk of development of colon and rectal cancer may be attributable to combined actions of environmental factors and endogenous promoting agents (Potter (1996)Eur. J. Cancer, 31A: 1033-1038). Thus, endogenous factors, which appear to be directly responsible for tumor cell growth, spreading and invasion (progression and metastasis) have been the focus of much attention in the pharmaceutical industry.
Among the endogenous factors that have been implicated in the development of colorectal cancer is nitric oxide. Nitric oxide is produced endogenously as a consequence of arginine metabolism by the family of nitric oxide synthase enzymes, which possess a wide range of physiological and pathophysiological actions (Moncada et al. (1991) Pharmacol. Rev., 43: 109-142).
Only one distinct form of nitric oxide synthase, known as Ca+2-independent inducible nitric oxide synthase (iNOS), a 130 Kd protein, is expressed in response to pro-inflammatory agents. Inducible nitric oxide synthase produces high, but sustained concentrations of nitric oxide when compared to the low levels produced by the Ca+2-dependent neuronal and endothelial isoforms of the enzyme (Nathan et al. (1994) Cell, 78: 915-918; Forstermann et al. (1995) Naunyn.-Schmiedebergs. Arch. Pharmakol., 352: 351-364).
Studies suggest that iNOS may also play a role in tumor development (Rao et al. (2000) Carcinogenesis, 21: 617-621). Increased iNOS expression and/or activity was reported in human gynecological (Thomsen et al. (1994) Cancer Res., 54: 1352-1354), breast (Thomsen et al. (1995) Br. J. Cancer, 72: 41-44), and central nervous system (Cobbs et al. (1995) Cancer Res., 55: 727-730) tumors. In addition, human colon adenomas exhibit increased expression and activity of iNOS (Ambs et al. (1998) Cancer Res., 58: 334-341).
Studies in experimental models of colon cancer indicate that azoxymethane-induced colon tumors have higher expression and activity, or both higher expression and activity, of iNOS by comparison to levels found in adjacent colonic tissue (Rao et al. (1998) Proc. Am. Assoc. Cancer Res., 39: 197; Takahashi et al. (1997) Cancer Res., 57: 1233-1237).
Inducible nitric oxide synthase has also been shown to be involved in the regulation of COX-2, which is believed to play a substantial role in colon tumorigenesis (Landino et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 15069-15074). Together these observations suggest that an iNOS may play an important role in colon tumorigenesis. In this connection, it is noteworthy that an iNOS-selective inhibitor, PBIT, prevented azoxymethane-induced colonic aberrant crypt foci development and iNOS activity in laboratory rodents (Rao et al. (1998) Proc. Am. Assoc. Cancer Res., 39: 197).
Also among the endogenous factors that have been implicated in the development of colorectal cancer are the cyclooxygenase (COX) enzymes. The involvement of COX enzymes in colorectal cancer has been revealed in work that has established the inhibitory effect of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, piroxicam and sulindac on colon cancer development. The collective results of epidemiological and animal studies present an inverse relationship between the use of NSAIDs and colon cancer (Thun et al. (1991) N. Engl. J. Med., 325: 1593-1596).
Additional evidence suggests that colon tumor inhibition by NSAIDs may be mediated through the modulation of arachidonic acid metabolism via cyclooxygenase enzymes which, in turn, inhibits immune responsiveness (Marnett (1992) Cancer Res., 52: 5575-5589; Smith (1992) Am. J. Physiol., 263: F181-F191).
However, cyclooxygenase enzymes include both a constitutive form, COX-1, which has a protective effect in the gastrointestinal system, and COX-2, which is an inducible form of the enzyme that is involved in pain, inflammation, edema, angiogenesis and tumor-related processes. Since NSAIDs do not selectively inhibit COX-2, prolonged administration of NSAIDs at therapeutic doses can cause gastrointestinal bleeding and ulceration, and also renal toxicity by blocking the activity of constitutive COX-1 activity.
Thus, NSAIDs probably render chemopreventive effects by blocking COX-2, but also produce adverse side effects by blocking the activity of COX-1. A need therefore existed for selective COX-2 inhibitors that act on inducible COX-2 enzyme but spare COX-1 activity and normal physiological functions of this enzyme. COX-2 selective inhibitors have since been developed, including, for example, celecoxib, rofecoxib, valdecoxib, parecoxib and related compounds. Some COX-2 selective inhibitors appear to have tumor-suppressive effects. The COX-2 selective inhibitor celecoxib has been shown to suppress induction of the colonic aberrant crypt foci (ACF) by azoxymethane and inhibit colon tumor formation (Reddy et al. (1996) Cancer Res., 56: 4566-4569; Kawamori et al. (1998) Cancer Res., 58: 409-412).
In addition, COX-2 inhibitors such as nemisulide and nabumetone inhibit formation of colonic aberrant crypt foci in male F344 rats (Rao et al. (1999) Proc. Am. Assoc. Cancer Res., 40: 373).
Additional evidence supporting a tumor-suppressive role for COX-2 comes from studies showing that MF-Tricyclic, a COX-2 inhibitor, blocks intestinal tumorigenesis in APCΔ716 mice (Oshima et al. (1996) Cell, 87: 803-809).
Based on preclinical and clinical efficacy studies, celecoxib has been approved for the treatment of patients with familial adenomatous polyps, a precancerous condition that precedes colon cancer in certain individuals. Additional recent studies on COX-2 have greatly improved the understanding of its role in colorectal cancer and other diseases (Taketo (1998) J. Natl. Cancer Inst., 90: 1609-1620). However, mechanistic studies support the hypothesis that COX-2 regulation is highly complex and influenced by various exogenous and endogenous factors.
Against this background, increasing interest has developed in finding combinations of low doses of two or more chemopreventive agents, each with different modes of action, so that overall treatment efficacy is improved while toxicity and adverse side effects of each agent are minimized. In this regard, preliminary work has examined a combination of an NSAID and an omithine decarboxylase inhibitor. More specifically, preliminary data suggests that lowest dose levels of piroxicam (an NSAID), and difluoromethylomithine (an omithine decarboxylase inhibitor) administered together are more effective in inhibiting colon tumorigenesis than when these agents are given individually even at higher levels (Reddy et al. (1990) Cancer Res., 50: 2562-2568).
A need however continues for new and effective combinations, and particularly for combinations that exploit the advantages of COX-2 selective inhibitors over NSAIDs.
Additional evidence suggests that aberrant crypt foci are especially suitable for use as a model system in which to test proposed chemopreventive combinations, and particularly those including COX-2 selective inhibitors. Aberrant crypt foci are recognized as early preneoplastic lesions and have consistently been observed in experimentally-induced colon carcinogenesis in laboratory animals (McLellan et al. (1991) Cancer Res., 51: 5270-5274).
Additional work has shown the presence of such lesions in the colonic mucosa of patients with colon cancer and have suggested that aberrant crypts are putative precursor lesions from which adenomas and carcinomas develop in the colon (Pretlow et al. (1992) J. Cell. Biochem., 16G (Suppl.): 55-62).
Aberrant crypt foci express mutations in the apc gene and ras oncogene that appear to be biomarkers of colon cancer development (Jen et al. (1994) Cancer Res., 54: 5523-5526).
Other preliminary studies indicate that both COX-2 and iNOS are over-expressed in the azoxymethane-induced colonic aberrant crypt foci (Rao et al. (1999) Proc. Am. Assoc. Cancer Res., 40: 373; Rao et al. (1998) Proc. Am. Assoc. Cancer Res., 39: 197). Additional evidence indicates that several inhibitors of aberrant crypt foci development reduce colon tumorigenesis in laboratory animals (Rao et al. (1995) Cancer Res., 55: 2310-2315; Rao et al. (1993) Cancer Res., 53: 4182-4188).
Thus, while COX-2 and iNOS have been separately implicated in tumor growth and development, known methods of treating and preventing tumor growth do not include the use of novel combinations of COX-2 selective inhibitors and iNOS-selective inhibitors. It would therefore be advantageous to find and describe new combinations of chemopreventive agents that allow reduced dosages of individual chemopreventive agents while maintaining or improving efficacy of the agents in the prophylaxis and treatment of cancers.